Fungal microbiota in newborn infants with and without respiratory distress syndrome.

BACKGROUND: Pneumocytis jirovecii infection in preterm newborns has recently been associated with neonatal respiratory distress syndrome and bronchopulmonary dysplasia. Changes in the bacterial microbiota of the airways have also been described in infants with bronchopulmonary dysplasia. However, until now there has been no information on the airway mycobiota in newborns. The purpose of this study was to describe the airway mycobiota in term and preterm newborns and its possible association with respiratory distress syndrome. METHODS: Twenty-six matched preterm newborns with and without respiratory distress syndrome were studied, as well as 13 term babies. The identification of the fungal microbiota was carried out using molecular procedures in aspirated nasal samples at birth. RESULTS: The ascomycota phylum was identified in 89.7% of newborns, while the basidiomycota phylum was found in 33.3%. Cladosporium was the predominant genus in both term and preterm infants 38.4% vs. 73% without statistical differences. Candida sake and Pneumocystis jirovecii were only found in preterm infants, suggesting a potential relationship with the risk of prematurity. CONCLUSIONS: This is the first report to describe the fungal microbiota of the airways in term and preterm infants with and without respiratory distress syndrome. Although no differences have been observed, the number of cases analyzed could be small to obtain conclusive results, and more studies are needed to understand the role of the fungal microbiota of the airways in neonatal respiratory pathology.

Biophysical evaluation of antiparallel triplexes for biosensing and biomedical applications.

Polypyrimidine sequences can be targeted by antiparallel clamps forming triplex structures either for biosensing or therapeutic purposes. Despite its successful implementation, their biophysical properties remain to be elusive. In this work, PAGE, circular dichroism and multivariate analysis were used to evaluate the properties of PPRHs directed to SARS-CoV-2 genome. Several PPRHs designed to target various polypyrimidine sites within the viral genome were synthesized. These PPRHs displayed varying binding affinities, influenced by factors such as the length of the PPRH and its GC content. The number and position of pyrimidine interruptions relative to the 4 T loop of the PPRH was found a critical factor, affecting the binding affinity with the corresponding target. Moreover, these factors also showed to affect in the intramolecular and intermolecular equilibria of PPRHs alone and when hybridized to their corresponding targets, highlighting the polymorphic nature of these systems. Finally, the functionality of the PPRHs was evaluated in a thermal lateral flow sensing device showing a good correspondence between their biophysical properties and detection limits. These comprehensive studies contribute to the understanding of the critical factors involved in the design of PPRHs for effective targeting of biologically relevant genomes through the formation of triplex structures under neutral conditions.

Fungal antigenic variation using mosaicism and reassortment of subtelomeric genes' repertoires.

Surface antigenic variation is crucial for major pathogens that infect humans. To escape the immune system, they exploit various mechanisms. Understanding these mechanisms is important to better prevent and fight the deadly diseases caused. Those used by the fungus Pneumocystis jirovecii that causes life-threatening pneumonia in immunocompromised individuals remain poorly understood. Here, though this fungus is currently not cultivable, our detailed analysis of the subtelomeric sequence motifs and genes encoding surface proteins suggests that the system involves the reassortment of the repertoire of ca. 80 non-expressed genes present in each strain, from which single genes are retrieved for mutually exclusive expression. Dispersion of the new repertoires, supposedly by healthy carrier individuals, appears very efficient because identical alleles are observed in patients from different countries. Our observations reveal a unique strategy of antigenic variation. They also highlight the possible role in genome rearrangements of small imperfect mirror sequences forming DNA triplexes.

Characteristics of clinical trials of influenza and respiratory syncytial virus registered in ClinicalTrials.gov between 2014 and 2021.

The randomized clinical trial (RCT) is the ideal and mandatory type of study to verify the effect and safety of a drug. Our aim is to examine the fundamental characteristics of interventional clinical trials on influenza and respiratory syncytial virus (RSV). This is a cross-sectional study of RCTs on influenza and RSV in humans between 2014 and 2021 registered in ClinicalTrials.gov. A total of 516 studies were identified: 94 for RSV, 423 for influenza, and 1 for both viruses. There were 51 RCTs of RSV vaccines (54.3%) and 344 (81.3%) for influenza virus vaccines (p < 0.001). Twelve (12.8%) RCTs for RSV were conducted only with women, and 6 were conducted only with pregnant women; for RCTs for influenza, 4 (0.9%) and 3, respectively. For RSV, 29 (31%) of the RCTs were exclusive to people under 5 years of age, and 21 (5%) for influenza virus (p < 0.001). For RSV, there are no RCTs exclusively for people older than or equal to 65 years and no phase 4 trials. RCTs on influenza virus and RSV has focused on vaccines. For the influenza virus, research has been consolidated, and for RSV, research is still in the development phase and directed at children and pregnant women.

The Impact of the Second Wave of the COVID-19 Pandemic on Non-COVID Hospital Care in a Tertiary Hospital in Spain.

In 2020, Spain ranked fourth among European countries with the highest excess mortality due to COVID-19 disease. This study evaluates the impact of the COVID-19 pandemic on non-COVID patients in a tertiary hospital during the second pandemic wave in Spain (22 June 2020-6 December 2020). Data from Virgen del Rocío University Hospital in Seville during that timeframe were compared with the data from the same period in the preceding two years (2018-2019). Between-group comparisons were performed using the Chi-squared test, Student's t-test, or Mann-Whitney U tests, as appropriate. A total of 63,137 non-COVID patients were included in this study. During the second pandemic wave, a 19% decrease was observed in the annual number of non-COVID admissions overall (18,260 vs. 22,439, p < 0.001), but a 10% increase in the proportion of emergency admissions (60.6% vs. 54.93%, p < 0.001), a higher severity level of patients (1.79 vs. 1.72, p < 0.001), a longer in-hospital stay (7.02 vs. 6.74 days, p < 0.001), a 26% increase in non-COVID mortality (4.9% vs. 3.9%, p < 0.001), and a 50% increase in global mortality (5.9 vs. 3.9, p < 0.001) were also observed. In terms of both medical and surgical diagnoses, a significant reduction in the number of admissions and an increase in in-hospital mortality were observed. These results demonstrate the significant impact of the pandemic on hospital care, similar to what was previously observed during the initial wave in the same hospital. Our findings emphasize the need to include non-COVID patients when assessing the broad impact of the pandemic on healthcare, beyond its direct effects on COVID-19 patients.

Impact of the first wave of the COVID-19 pandemic on non-COVID inpatient care in southern Spain.

We assessed the impact of the first wave of COVID-19 pandemic on non-COVID hospital admissions, non-COVID mortality, factors associated with non-COVID mortality, and changes in the profile of non-COVID patients admitted to hospital. We used the Spanish Minimum Basic Data Set with diagnosis grouped according to the Diagnostic Related Groups. A total of 10,594 patients (3% COVID-19; 97% non-COVID) hospitalised during the first wave in 2020 (27-February/07-June) were compared with those hospitalised within the same dates of 2017-2019 (average annual admissions: 14,037). We found a decrease in non-COVID medical (22%) and surgical (33%) hospitalisations and a 25.7% increase in hospital mortality among non-COVID patients during the first pandemic wave compared to pre-pandemic years. During the officially declared sub-period of excess mortality in the area (17-March/20-April, in-hospital non-COVID mortality was even higher (58.7% higher than the pre-pandemic years). Non-COVID patients hospitalised during the first pandemic wave (compared to pre-pandemic years) were older, more frequently men, with longer hospital stay and increased disease severity. Hospitalisation during the first pandemic wave in 2020, compared to hospitalisation during the pre-pandemic years, was an independent risk factor for non-COVID mortality (HR 1.30, 95% CI 1.07-1.57, p = 0.008), reflecting the negative impact of the pandemic on hospitalised patients.

Fungal microbiota dynamics and its geographic, age and gender variability in patients with cystic fibrosis.

OBJECTIVES: In cystic fibrosis (CF), there is a predisposition to bronchial colonization by potentially pathogenic microorganisms, such as fungi. Our aims were to describe the dynamics of respiratory mycobiota in patients with CF and to evaluate the geographic, age and gender variability in its distribution. METHODS: Cohort study in which 45 patients with CF from four hospitals in three Spanish cities were followed up during a 1-year period, obtaining spontaneous sputum samples every 3 to 6 months. Fungal microbiota were characterized by Internal Transcribed Spacer sequencing and Pneumocystis jirovecii was identified by nested PCR in a total of 180 samples. RESULTS: The presence of fungi were detected in 119 (66.11%) of the 180 samples and in 44 (97.8%) of the 45 patients: 19 were positive and 1 negative throughout all follow-ups and the remaining 25 presented alternation between positive and negative results. A total of 16 different genera were identified, with Candida spp. (50/180, 27.78%) and Pneumocystis spp. (44/180, 24.44%) being the most prevalent ones. The distribution of fungal genera was different among the evaluated centres (p < 0.05), by age (non-adults aged 6-17 years vs. adults aged ≥18 years) (p < 0.05) and by gender (p < 0.05). DISCUSSION: A high prevalence of fungal respiratory microbiota in patients with CF was observed, whose dynamics are characterized by the existence of multiple cycles of clearance and colonization, reporting the existence of geographic, age and gender variability in the distribution of fungal genera in this disease.

Genetic and Morphological Identification of Spirometra decipiens in Snakes and Domestic Dog Found in Cuba.

Spirometra (Cestoda: Diphyllobothriidea) affects humans and some species of domestic and wild animals which eventually interact with humans. In this article, we report three new cases of Spirometra decipiens (Diesing, 1850) infection observed in two intermediate hosts and one definitive host, in Cuba. Genetic and morphological identification of S. decipiens in two snakes and a domestic dog were carried out by molecular means and routine histological study using hematoxylin-eosin staining, respectively. Taken together, the anatomical location, the host species infected with the specimens and their morphological and genetic features, all the samples were identified as S. decipiens. In each of the three cases, PCR assays using specific primers amplified bands that corresponded to S. decipiens species. To our knowledge, this paper is the first report of S. decipiens in species of Cuban endemic fauna and in the Caribbean islands. These species constitute a real or potential risk of transmission of Spirometra to humans in Cuba.

Nonspecific Effects of Infant Vaccines Make Children More Resistant to SARS-CoV-2 Infection.

A myriad of reasons, or a combination of them, have been alluded to in order to explain the lower susceptibility of children to SARS-CoV-2 infection and the development of severe forms of COVID-19. This document explores an additional factor, still little addressed in the medical literature related to the matter: nonspecific resistance to SARS-CoV-2 that could be generated by vaccines administered during childhood. The analysis carried out allows one to conclude that a group of vaccines administered during childhood is associated with a lower incidence and severity of SARS-CoV-2 infection among pediatric ages. Looking from an epidemiological perspective, this conclusion must be taken into consideration in order to ensure greater rationality in the design and implementation of prevention and control actions, including the administration of the COVID-19 vaccine, for these ages.

Overlapping of Pulmonary Fibrosis of Postacute COVID-19 Syndrome and Tuberculosis in the Helminth Coinfection Setting in Sub-Saharan Africa.

There is an increasing attention to the emerging health problem represented by the clinical and functional long-term consequences of SARS-CoV-2 infection, referred to as postacute COVID-19 syndrome. Clinical, radiographic, and autopsy findings have shown that a high rate of fibrosis and restriction of lung function are present in patients who have recovered from COVID-19. Patients with active TB, or those who have recovered from it, have fibrotic scarred lungs and, consequently, some degree of impaired respiratory function. Helminth infections trigger predominantly type 2 immune responses and the release of regulatory and fibrogenic cytokines, such as TGF-ß. Here, we analyze the possible consequences of the overlapping of pulmonary fibrosis secondary to COVID-19 and tuberculosis in the setting of sub-Saharan Africa, the region of the world with the highest prevalence of helminth infection.

Novel genes and sex differences in COVID-19 severity.

Here, we describe the results of a genome-wide study conducted in 11 939 coronavirus disease 2019 (COVID-19) positive cases with an extensive clinical information that were recruited from 34 hospitals across Spain (SCOURGE consortium). In sex-disaggregated genome-wide association studies for COVID-19 hospitalization, genome-wide significance (P < 5 × 10-8) was crossed for variants in 3p21.31 and 21q22.11 loci only among males (P = 1.3 × 10-22 and P = 8.1 × 10-12, respectively), and for variants in 9q21.32 near TLE1 only among females (P = 4.4 × 10-8). In a second phase, results were combined with an independent Spanish cohort (1598 COVID-19 cases and 1068 population controls), revealing in the overall analysis two novel risk loci in 9p13.3 and 19q13.12, with fine-mapping prioritized variants functionally associated with AQP3 (P = 2.7 × 10-8) and ARHGAP33 (P = 1.3 × 10-8), respectively. The meta-analysis of both phases with four European studies stratified by sex from the Host Genetics Initiative (HGI) confirmed the association of the 3p21.31 and 21q22.11 loci predominantly in males and replicated a recently reported variant in 11p13 (ELF5, P = 4.1 × 10-8). Six of the COVID-19 HGI discovered loci were replicated and an HGI-based genetic risk score predicted the severity strata in SCOURGE. We also found more SNP-heritability and larger heritability differences by age (<60 or ≥60 years) among males than among females. Parallel genome-wide screening of inbreeding depression in SCOURGE also showed an effect of homozygosity in COVID-19 hospitalization and severity and this effect was stronger among older males. In summary, new candidate genes for COVID-19 severity and evidence supporting genetic disparities among sexes are provided.

Pneumocystis primary infection in non-immunosuppressed infants in Lima, Peru.

OBJECTIVES: To provide original data on Pneumocystis primary infection in non-immunosuppressed infants from Peru. METHODS: A cross sectional study was performed. Infants less than seven months old, without any underlying medical conditions attending the "well baby" outpatient clinic at one hospital in Lima, Peru were prospectively enrolled during a 15-month period from November 2016 to February 2018. All had a nasopharyngeal aspirate (NPA) for detection of P. jirovecii DNA using a PCR assay, regardless of respiratory symptoms. P. jirovecii DNA detection was considered to represent pulmonary colonization contemporaneous with Pneumocystis primary infection. Associations between infants' clinical and demographic characteristics and results of P. jirovecii DNA detection were analyzed. RESULTS: P. jirovecii DNA was detected in 45 of 146 infants (30.8%) and detection was not associated with concurrent respiratory symptoms in 40 of 45 infants. Infants with P. jirovecii had a lower mean age when compared to infants not colonized (p <0.05). The highest frequency of P. jirovecii was observed in 2-3-month-old infants (p < 0.01) and in the cooler winter and spring seasons (p <0.01). Multivariable analysis showed that infants living in a home with ≤ 1 bedroom were more likely to be colonized; Odds Ratio =3.03 (95%CI 1.31-7.00; p = 0.01). CONCLUSION: Pneumocystis primary infection in this single site in Lima, Peru, was most frequently observed in 2-3-month-old infants, in winter and spring seasons, and with higher detection rates being associated with household conditions favoring close inter-individual contacts and potential transmission of P. jirovecii.

Multilocus Genotyping of Pneumocystis jirovecii from Deceased Cuban AIDS Patients Using Formalin-Fixed and Paraffin-Embedded Tissues.

The results of the genotypic characterization of Pneumocystis jirovecii are described in lung tissue samples from 41 Cubans who died of AIDS with pneumocystosis between 1995 and 2008. Histological sections of the lung preserved as formalin-fixed and paraffin-embedded tissue were examined. PCR amplification and nucleotide sequencing of the two mitochondrial genes (large and small) of the pathogen allowed verification of a predominance of genotype 3 (85T/248C) of the large mitochondrial gene and genotype 3 (160A/196T) of the small mitochondrial gene over a period of 14 years (1995-2008). These results suggest that the 85T/248C//160A/196T genotype circulates with the highest frequency (81.3%) among AIDS patients in Cuba. Multilocus analysis indicates a limited circulation of pathogen genotypes on the island with the existence of a clonal genotype with an epidemic structure. Furthermore, it appears that circulating strains of P. jirovecii have not developed mutations related to sulfonamide resistance. Taken together, the data in this study revealed important elements about pneumocystosis in Cuban patients dying of AIDS and the usefulness of formalin-fixed and paraffin-embedded samples to carry out molecular epidemiology studies of P. jirovecii.

Update on Dihydropteroate Synthase (DHPS) Mutations in Pneumocystis jirovecii.

A Pneumocystis jirovecii is one of the most important microorganisms that cause pneumonia in immunosupressed individuals. The guideline for treatment and prophylaxis of Pneumocystis pneumonia (PcP) is the use of a combination of sulfa drug-containing trimethroprim and sulfamethoxazole. In the absence of a reliable method to culture Pneumocystis, molecular techniques have been developed to detect mutations in the dihydropteroate synthase gene, the target of sulfa drugs, where mutations are related to sulfa resistance in other microorganisms. The presence of dihydropteroate synthase (DHPS) mutations has been described at codon 55 and 57 and found almost around the world. In the current work, we analyzed the most common methods to identify these mutations, their geographical distribution around the world, and their clinical implications. In addition, we describe new emerging DHPS mutations. Other aspects, such as the possibility of transmitting Pneumocystis mutated organisms between susceptible patients is also described, as well as a brief summary of approaches to study these mutations in a heterologous expression system.

It is still PCP that can stand for Pneumocystis pneumonia: Appeal for generalized use of only one acronym.

Twenty-years ago, considering the host specificity of Pneumocystis species, the human-derived Pneumocystis, Pneumocystis carinii formae specialis hominis, was renamed Pneumocystis jirovecii. Pneumocystis carinii formae specialis carinii was finally renamed Pneumocystis carinii and kept for the species derived from Rattus norvegicus. P. jirovecii is now widely used by most authors. The PCP acronym that initially referred to "Pneumocystis cariniipneumonia" was contemporaneously redefined to stand for Pneumocystispneumonia in order to avoid changing the acronym of the name of the disease that clinicians have used for several decades. Using analysis of multidata bases on PubMed, we have noted a recent acceleration in the use of PJP for Pneumocystis jiroveciipneumonia, which may be grammatically correct but not in accordance with retaining PCP, which was proposed in the early 2000s. Through this reminder, in order to standardize the literature on P. jirovecii, we plead for the use of only one acronym, PCP. LAY SUMMARY: Through this reminder on Pneumocystis nomenclature, we plead for the use of only one acronym, PCP, the retention of which was proposed in the early 2000s, and which currently stands for Pneumocystispneumonia.

Virulence Plasmids of Rhodococcus equi Isolates From Cuban Patients With AIDS.

Rhodococcus equi is an animal pathogen and zoonotic human opportunistic pathogen associated with immunosuppressive conditions. The pathogenicity of R. equi is linked to three animal host-associated virulence plasmids encoding a family of "Virulence Associated Proteins" (VAPs). Here, the PCR-based TRAVAP molecular typing system for the R. equi virulence plasmids was applied to 26 R. equi strains isolated between 2010 and 2016 at the Institute of Tropical Medicine "Pedro Kourí," Cuba, from individuals living with HIV/AIDS. TRAVAP detects 4 gene markers, traA common to the three virulence plasmids, and vapA, vapB, and vapN specific to each of the host-associated plasmid types (equine pVAPA, porcine pVAPB, and ruminant pVAPN). Of the 26 isolates, six were positive to the vapB (porcine-type) marker, 4 (15.4%) to the vapA (equine-type) marker, and 1 (3.8%) to the vapN (ruminant-type) marker. Most of the isolates 14 (53.8%) were negative to all TRAVAP markers, suggesting they lacked a virulence plasmid. To our knowledge, this work is the first to report the molecular characterization of R. equi isolates from Cuba. Our findings provide insight into the zoonotic origin of R. equi infections in people and the potential dispensability of the virulence plasmid in immunosuppressed patients.

Pneumocystis jirovecii among patients with cystic fibrosis and their household members.

We conducted a pilot study of patients with cystic fibrosis (CF) to assess intra-family transmission of P. jirovecii and compare it with data on other prevalent pathogens such as P. aeruginosa and S. pneumoniae, in which respiratory transmission has already been documented. Oral swab samples from 10 patients with CF and 15 household members were collected at baseline and 2 weeks later. P. aeruginosa and S. pneumoniae were assessed using standardized culture methods and PCR, and P. jirovecii was assessed using real and nested PCR, genotyping the positive samples by direct sequencing. P. aeruginosa cultures were positive for 7/10 (70%) of patients with CF at baseline and was identified by PCR in 8/10 (80%) of cases at baseline and 2 weeks later. S. pneumoniae cultures were negative for all patients, but the microorganism was identified by PCR in two cases. P. jirovecii was detected by real time and nested PCR in 5/10 (50%) of the patients at the two time points. In the household members, P. aeruginosa and P. jirovecii were identified in 7/15 (46.7%), and S. pneumoniae was identified in 8/15 (53,3%). The concordance of positive or negative pairs of patients with CF and their household members was 33.3% (5/15) for P. aeruginosa, 46.7% (7/15) for S. pneumonia and 93.3% (14/15) for P. jirovecii. The concordance for P. jirovecii genotypes among five pairs with available genotype was 100%. This study suggests for the first time the possible transmission of Pneumocystis in the home of patients with CF, indicating that patients and their household members are reservoirs and possible sources of infection. LAY SUMMARY: This study suggests for the first time the possible transmission of Pneumocystis in the family environment of patients with cystic fibrosis, indicating that patients and their household members are reservoirs and possible sources of this infection.

Prevalence of Pneumocystosis in Sub-Saharan Africa and Helminth Immune Modulation.

Sub-Saharan Africa is the region of the world with the highest prevalence of helminth infections. To protect themselves from the defensive mechanisms of their respective hosts, helminths modulate their immune responses. This modulation has relevant clinical and epidemiological consequences, including the inhibition of inflammatory processes that characterize infection by other microorganisms. Severe Pneumocystis pneumonia is characterized by an intense inflammatory reaction that can lead to death. Acquired immunodeficiency syndrome is the main predisposing factor to the development of pneumocystosis. Although the introduction of highly active antiretroviral therapy has led to a notable decline in the incidence of acquired immunodeficiency syndrome-associated complications, pneumocystosis continues to be an important global health problem. Despite the high incidence of human immunodeficiency virus infection in the sub-Saharan region, the prevalence of Pneumocystis pneumonia there has been lower than expected. Several factors, or combinations thereof, may contribute to this evolution. Here, we hypothesize the possible role of helminth immune modulation as an important issue at play. On the other hand, and looking ahead, we believe that the immune modulation achieved by helminths may be an important factor to consider during the design and evaluation processes of vaccines against Pneumocystis jirovecii to be used in Sub-Saharan Africa. The requirements of a balanced triggering of different types of immune responses for controlling the infection produced by this microorganism, as observed during experiments in animal models, support this final consideration.

A Real-Time PCR Assay for Detection of Low Pneumocystis jirovecii Levels.

Here we report a new real-time PCR assay using SYBR Green which provides higher sensitivity for the specific detection of low levels of Pneumocystis jirovecii. To do so, two primer sets were designed, targeting the family of genes that code for the most abundant surface protein of Pneumocystis spp., namely the major surface glycoproteins (Msg), and the mitochondrial large subunit rRNA (mtLSUrRNA) multicopy gene, simultaneously detecting two regions. PCR methods are instrumental in detecting these low levels; however, current nested-PCR methods are time-consuming and complex. To validate our new real-time Msg-A/mtLSUrRNA PCR protocol, we compared it with nested-PCR based on the detection of Pneumocystis mitochondrial large subunit rRNA (mtLSUrRNA), one of the main targets used to detect this pathogen. All samples identified as positive by the nested-PCR method were found positive using our new real-time PCR protocol, which also detected P. jirovecii in three nasal aspirate samples that were negative for both rounds of nested-PCR. Furthermore, we read both rounds of the nested-PCR results for comparison and found that some samples with no PCR amplification, or with a feeble band in the first round, correlated with higher Ct values in our real-time Msg-A/mtLSUrRNA PCR. This finding demonstrates the ability of this new single-round protocol to detect low Pneumocystis levels. This new assay provides a valuable alternative for P. jirovecii detection, as it is both rapid and sensitive.